Three dimensional protein structures have extremely high commercial value since they allow for the use of rational (structure-based) design and engineering of novel drug molecules that bind to the protein of interest. Furthermore, they facilitate the rational engineering of novel proteins with desired properties. One method of protein X-ray crystallographic structure determination involves: (1) preparation of purified protein; (2) crystallization of the protein; (3) isolation and alignment of single protein crystals in front of an intense and focused X-ray beam; (4) collection of complete X-ray diffraction data sets by rotating the single crystal within the X-ray beam; (5) capturing the diffraction spots on a recording device that measures X-ray spot position and intensity; (6) computational analysis of the X-ray diffraction data to derive experimental electron density maps of the crystal. These maps are in turn used to derive a three dimensional chemical model of the protein that formed the crystal. However, a general problem in the use of X-ray diffraction methods to determine the three-dimensional structures of proteins at near atomic resolution is the rate-limiting step of protein crystallization.
Membrane proteins are a broad class of proteins which bind to and/or traverse a lipid bilayer (membrane) that surrounds all living cells. Membrane proteins are typically involved in the controlled movement of substances and/or signals across the cell membrane. In so doing, membrane proteins enable rapid communication between the inside and outside of living cells. Examples of membrane proteins include ion channels, signaling receptors, hormone receptors, light receptors, and adhesion proteins. Such membrane proteins are the targets of several blockbuster drugs on the market as well as a variety of drugs under development at pharmaceutical companies to treat numerous aliments.
Historically, membrane proteins have been notoriously difficult to crystallize. This is due to their hydrophobic (water hating) and/or lipophilic (fat loving) nature which makes them difficult to purify in large quantity and reduces their overall solubility in aqueous solutions. These factors make it difficult to crystallize membrane protein since they tend to be unstable at concentration in aqueous solutions that are required for the nucleation of crystal growth by crystallization methods used for soluble (non-membrane bound) proteins.
In 1996, Landau and Rosenbusch described the novel use of Lipidic Cubic Phases for the crystallization of membrane proteins. According to this method, detergent solubilized membrane protein is mixed with monoolein (or monopalmitolein) and water (or buffered solutions), followed by multiple rounds of centrifugation. This extensive method allowed for gentle mixing of the materials over 2 to 3 hours to create a viscous, bicontinuous cubic phase, a cured lipid bilayer, extending in three dimensions and permeated by aqueous channels. The membrane proteins can partition into the lipid bilayer and can diffuse in three dimensions which allows them to explore many potential spatial packing configurations that can lead to crystal growth of the protein within lipidic mesophases, such as the so called “Lipidic Cubic Phase” (LCP).
The Landau and Rosenbusch original LCP crystallization method involves the use of small glass vials into which monoolein, protein and buffered water are added, followed by multiple centrifugations to create the LCP. After the LCP is created, small quantities of dry salt are added and the vials are sealed and incubated. Crystal growth is monitored by examining each glass vial under a stereo microscope. This original lipidic mesophase protocol is tedious, time consuming, and requires more initial protein material than the amount that is necessary for conventional crystallization based on vapor diffusion. The addition of dry salt is time consuming, in particular, as it requires a precision weighing step. In addition, the observation of crystal growth is tedious since it involves multiple tube handling events. Because of these limitations the Landau and Rosenbusch LCP method has generally not been put to use by the protein crystallography community.